1. Field of the Invention
The invention is directed to a multispectral illumination device for a microscope or for a reader.
2. Description of Related Art
An important application for illumination devices is in arrangements for imaging and examination which are provided for generating images of an object or sample to be examined. Typical examples of such imaging devices are microscopes, particularly microscopes with widefield optics which image a given area of the sample to be imaged or examined. Fluorescence examination plays a special role in the optical examination of samples. In this case, the sample is irradiated by an excitation beam with a suitable excitation spectrum which is selected depending on one or more fluorescent dyes. When these fluorescent dyes are found in the sample, they interact with the excitation radiation and emit fluorescent radiation that is characteristic for the dye. Detection of the sample is made possible in this way. Not only the presence of fluorescent dyes but their concentration and spatial arrangement can also be determined.
Nanoparticles, for example, quantum dots, or other dyes which fluoresce in at least one wavelength can also be used instead of fluorescent dyes.
Light sources such as mercury arc lamps or xenon arc lamps are conventionally used in order to excite many different fluorescent dyes or nanoparticles. These light sources have a broad emission spectrum which extends from ultraviolet to near-infrared. The selection of the excitation spectrum required for a dye is usually carried out in microscopes through a set of filters, a so-called excitation filter, a beamsplitter, and an emission filter. Excitation filters and beamsplitter filters are selected in such a way that they pass or reflect the portion of the spectrum of the light source that is required for exciting the dye in the sample. The fluorescent radiation which is then emitted by the sample passes through the beamsplitter and the emission filter, which causes a further suppression of scattered excitation light. This combination of filters provides for improved contrast in a fluorescence image. Depending on the arrangement of the beam path in the microscope, the beamsplitter can also be omitted.
Often, filter sets with only one wavelength band are used in fluorescence experiments to excite the fluorescent dye. However, there are also multi-bandpass filter sets which make it possible to observe a fluorescent sample in multiple colors simultaneously or to change spectral regions in rapid sequence. Accordingly, a multicolored impression of the sample can be generated in the experiment, which allows different parts of the sample to be highlighted in color simultaneously. At the present time, multi-bandpass filter sets with two, three, or four transmission bands are available for fluorescence microscopy. These filters are called double bandpass filters, triple bandpass filters and quad bandpass filters. Triple bandpass filters and quad bandpass filters in particular are used, e.g., in FISH (fluorescence-in-situ hybridization) experiments. In such experiments it is desirable to be able to change quickly between all spectral regions of the light source or for all spectral regions of the light source to be available simultaneously.
More recently, light emitting diodes (LEDs) have been used for illumination in fluorescence microscopy. They have a substantially longer lifetime than arc lamps and offer optical emission outputs comparable to those of arc lamps in some spectral regions. Additional advantages are that they generate less heat, have higher electrical to optical efficiencies, faster switching speed and a narrower emission spectrum. Fast switching speeds of the illumination device are advantageous, for example, in experiments in which a microscopic object is tracked and in experiments for fast measurement of concentrations of dyes (ratio imaging). Accordingly, for digital recording of processes, processes requiring image frequencies above 50 Hz can be imaged.
At present, high-power light emitting diodes are available in virtually all relevant wavelength ranges for fluorescence microscopy. An overview is given in “Handbook of biological confocal microscopy” (third edition, Springer 2006, pages 126 ff.). Currently, optical power outputs in the range of several 100 mW are already achieved, and the spectral half-intensity width is in the range of 5 nm to 40 nm.
At the current time, there are no high-power light emitting diodes available in the ranges from 405 nm to 445 nm and 550 nm to 580 nm, in particular.
Typical spectral half-intensity widths of the spectral curves (absorption and emission) of fluorescent dyes are typically around 30 nm.
With regard to simultaneous multicolored illumination of fluorescent samples by a plurality of LEDs, the spatial and spectral coupling of the light radiation emitted by the respective LEDs is particularly important. Because of the narrow emission spectra of the LEDs, diffractive optics are also suitable for this purpose. With spectral coupling by means of diffractive elements, broadband light sources would be spatially blurred because their dispersion is dependent upon wavelength.
US20050224692 describes a microscope with an illumination device having a plurality of arrangements for coupling different-colored LEDs. The coupling of the LEDs by a cemented multiple-prism, dichromatic mirrors, an individual prism, and a grating is described. The coupling of three LEDs with a fixed emission spectrum is described. However, since LEDs have narrow emission spectra, an arrangement of this kind covers only part of the spectrum for fluorescence experiments. The description does not address expanding the available spectral region of the illumination device by exchanging individual LEDs and, as the case may be, coupling optics for LEDs of another spectrum in a modular manner.
DE102005054184 describes a multispectral illumination device in which lighting means such as LEDs are coupled together in a treelike structure by dichromats. The unit comprising lighting means and associated means for beam shaping and spectral shaping (collimation and filtering) along with the mechanical mounting of these components will be referred to hereinafter as a lighting module. Without loss of generality, the lighting means themselves can in turn comprise a plurality of light sources, for example, an array of LEDs. Without loss of generality, let the spectral half-intensity width of an individual spectrum of a lighting module be less than 40 nm. Means arranged downstream for beam coupling of the lighting modules will be referred to hereinafter as coupling modules. The lighting modules in DE102005054184 preferably comprise at least one LED, a filter and collimating optics which serve for spectral shaping and beam shaping of the light radiation emitted by the LEDs.
The total spectrum of the illumination device is composed of the individual spectra of the lighting modules whose emission radiation outputs can be adjusted independently from one another. In particular, all of the individual spectra are available simultaneously at the output of the illumination device. As has already been described, light emitting diodes typically have spectral half-intensity widths in the range of 5 nm to 40 nm. To make use of the above-described advantages of LEDs in fluorescence microscopy, an LED-based multispectral illumination device would have to have a large quantity of different-colored LEDs which, together, would cover virtually the entire spectral range of visible and near-infrared light. Accordingly, at least ten different-colored LEDs would be required just for complete coverage of the visible spectral region from about 350 nm to 700 nm. Obviously, an illumination device comprising this many LEDs would be complicated and expensive to build. Further, the space requirement for this is large and space would be taken up in the laboratory. DE102005054184 suggests extending the tree structure in order to expand the spectral coverage. An expansion of this kind involves a more complicated design of the illumination device because more lighting modules and more coupling modules would have to be accommodated in the illumination device.
WO2006072886 describes an illumination device for a transmitted-light fluorescence microscope in which individual lighting modules comprising, respectively, an LED, optics and a filter are coupled into the microscope by a mechanically exchangeable beamsplitter in a coupling module. The lighting modules can be detached from the coupling module individually and exchanged. The described illumination device only allows two individual spectra of the three lighting modules to be coupled. In order to change to the third lighting module, the coupling module must be re-plugged. Accordingly, a maximum of two individual spectra of the lighting modules is available in the microscope at one time. An expansion to three or more lighting modules whose individual spectra are present simultaneously at the output of the illumination device is impossible by nature of the design.